Our objectives in this grant are to develop a detailed understanding of the concerted in vitro replication reaction which occurs upon mixing seven purified T4 bacteriophage DNA replication proteins, corresponding to genes 32, 41, 43, 44, 45, 61 and 62. Because of the complexity of replication enzymology, this requires study of a variety of "partial reactions", in which subsets of the seven replication proteins are mixed together with special DNA templates on which their individual activities can be seen. Eventually, we want to know exactly what each protein does in the replication reaction, including its detailed three-dimensional structure from X-ray crystallography, the structure of the multienzyme complex, and the rationale for the complexity of the replication process observed. Our specific aims for this grant period are encompassed by the following studies: 1. protein purification and the development of bacterial strains which vastly overproduce T4 gene 43, 41, 45 and 61 proteins: 2. quantitation of protein-protein and protein:DNA affinities; 3. characterization of the two DNA-dependent ATPase activities (the 44/62 and the 41 proteins); 4. characterization of incomplete reactions which polymerize DNA on single-stranded templates; 5. RNA primer synthesis, sequencing and the sites of de novo DNA chain starts; 6. the meeting and sealing of adjacent primed DNA pieces on the lagging strand; 7. the coupling of leading and lagging strand syntheses in the complete reaction on double-helical templates; 8. the source of the enormous templating fidelity observed in the complete reaction.